Publications by authors named "Jeffrey A Engelman"

177 Publications

SHP2 blockade enhances anti-tumor immunity via tumor cell intrinsic and extrinsic mechanisms.

Sci Rep 2021 Jan 14;11(1):1399. Epub 2021 Jan 14.

Oncology Disease Area, Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, MA, 02139, USA.

SHP2 is a ubiquitous tyrosine phosphatase involved in regulating both tumor and immune cell signaling. In this study, we discovered a novel immune modulatory function of SHP2. Targeting this protein with allosteric SHP2 inhibitors promoted anti-tumor immunity, including enhancing T cell cytotoxic function and immune-mediated tumor regression. Knockout of SHP2 using CRISPR/Cas9 gene editing showed that targeting SHP2 in cancer cells contributes to this immune response. Inhibition of SHP2 activity augmented tumor intrinsic IFNγ signaling resulting in enhanced chemoattractant cytokine release and cytotoxic T cell recruitment, as well as increased expression of MHC Class I and PD-L1 on the cancer cell surface. Furthermore, SHP2 inhibition diminished the differentiation and inhibitory function of immune suppressive myeloid cells in the tumor microenvironment. SHP2 inhibition enhanced responses to anti-PD-1 blockade in syngeneic mouse models. Overall, our study reveals novel functions of SHP2 in tumor immunity and proposes that targeting SHP2 is a promising strategy for cancer immunotherapy.
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http://dx.doi.org/10.1038/s41598-021-80999-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809281PMC
January 2021

LXH254, a Potent and Selective ARAF-Sparing Inhibitor of BRAF and CRAF for the Treatment of MAPK-Driven Tumors.

Clin Cancer Res 2021 Apr 22;27(7):2061-2073. Epub 2020 Dec 22.

Novartis Institutes for Biomedical Research, Cambridge, Massachusetts.

Purpose: Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, LXH254.

Experimental Design: LXH254 was profiled in biochemical, , and assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF.

Results: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and mutants, but had only modest activity in mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, , LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive.

Conclusions: LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2563DOI Listing
April 2021

TGFβ-blockade uncovers stromal plasticity in tumors by revealing the existence of a subset of interferon-licensed fibroblasts.

Nat Commun 2020 12 9;11(1):6315. Epub 2020 Dec 9.

Immuno-Oncology, Novartis Institutes for BioMedical Research, 250 Massachusetts Ave, Cambridge, MA, 02139, USA.

Despite the increasing interest in targeting stromal elements of the tumor microenvironment, we still face tremendous challenges in developing adequate therapeutics to modify the tumor stromal landscape. A major obstacle to this is our poor understanding of the phenotypic and functional heterogeneity of stromal cells in tumors. Herein, we perform an unbiased interrogation of tumor mesenchymal cells, delineating the co-existence of distinct subsets of cancer-associated fibroblasts (CAFs) in the microenvironment of murine carcinomas, each endowed with unique phenotypic features and functions. Furthermore, our study shows that neutralization of TGFβ in vivo leads to remodeling of CAF dynamics, greatly reducing the frequency and activity of the myofibroblast subset, while promoting the formation of a fibroblast population characterized by strong response to interferon and heightened immunomodulatory properties. These changes correlate with the development of productive anti-tumor immunity and greater efficacy of PD1 immunotherapy. Along with providing the scientific rationale for the evaluation of TGFβ and PD1 co-blockade in the clinical setting, this study also supports the concept of plasticity of the stromal cell landscape in tumors, laying the foundation for future investigations aimed at defining pathways and molecules to program CAF composition for cancer therapy.
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http://dx.doi.org/10.1038/s41467-020-19920-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725805PMC
December 2020

Combinations with Allosteric SHP2 Inhibitor TNO155 to Block Receptor Tyrosine Kinase Signaling.

Clin Cancer Res 2021 Jan 12;27(1):342-354. Epub 2020 Oct 12.

Oncology Disease Area, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.

Purpose: SHP2 inhibitors offer an appealing and novel approach to inhibit receptor tyrosine kinase (RTK) signaling, which is the oncogenic driver in many tumors or is frequently feedback activated in response to targeted therapies including RTK inhibitors and MAPK inhibitors. We seek to evaluate the efficacy and synergistic mechanisms of combinations with a novel SHP2 inhibitor, TNO155, to inform their clinical development.

Experimental Design: The combinations of TNO155 with EGFR inhibitors (EGFRi), BRAFi, KRASi, CDK4/6i, and anti-programmed cell death-1 (PD-1) antibody were tested in appropriate cancer models and , and their effects on downstream signaling were examined.

Results: In EGFR-mutant lung cancer models, combination benefit of TNO155 and the EGFRi nazartinib was observed, coincident with sustained ERK inhibition. In BRAF colorectal cancer models, TNO155 synergized with BRAF plus MEK inhibitors by blocking ERK feedback activation by different RTKs. In KRAS cancer cells, TNO155 effectively blocked the feedback activation of wild-type KRAS or other RAS isoforms induced by KRASi and greatly enhanced efficacy. In addition, TNO155 and the CDK4/6 inhibitor ribociclib showed combination benefit in a large panel of lung and colorectal cancer patient-derived xenografts, including those with KRAS mutations. Finally, TNO155 effectively inhibited RAS activation by colony-stimulating factor 1 receptor, which is critical for the maturation of immunosuppressive tumor-associated macrophages, and showed combination activity with anti-PD-1 antibody.

Conclusions: Our findings suggest TNO155 is an effective agent for blocking both tumor-promoting and immune-suppressive RTK signaling in RTK- and MAPK-driven cancers and their tumor microenvironment. Our data provide the rationale for evaluating these combinations clinically.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2718DOI Listing
January 2021

A Proof of Concept for Biomarker-Guided Targeted Therapy against Ovarian Cancer Based on Patient-Derived Tumor Xenografts.

Cancer Res 2020 10 3;80(19):4278-4287. Epub 2020 Aug 3.

Laboratory of Systems Pharmacology, Harvard Medical School, Boston, Massachusetts.

Advanced ovarian cancers are a leading cause of cancer-related death in women and are currently treated with surgery and chemotherapy. This standard of care is often temporarily successful but exhibits a high rate of relapse, after which, treatment options are few. Here we investigate whether biomarker-guided use of multiple targeted therapies, including small molecules and antibody-drug conjugates, is a viable alternative. A panel of patient-derived ovarian cancer xenografts (PDX), similar in genetics and chemotherapy responsiveness to human tumors, was exposed to 21 monotherapies and combination therapies. Three monotherapies and one combination were found to be active in different subsets of PDX. Analysis of gene expression data identified biomarkers associated with responsiveness to each of the three targeted therapies, none of which directly inhibits an oncogenic driver. While no single treatment had as high a response rate as chemotherapy, nearly 90% of PDXs were eligible for and responded to at least one biomarker-guided treatment, including tumors resistant to standard chemotherapy. The distribution of biomarker positivity in The Cancer Genome Atlas data suggests the potential for a similar precision approach in human patients. SIGNIFICANCE: This study exploits a panel of patient-derived xenografts to demonstrate that most ovarian tumors can be matched to effective biomarker-guided treatments.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-3850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541581PMC
October 2020

Resistance to allosteric SHP2 inhibition in FGFR-driven cancers through rapid feedback activation of FGFR.

Oncotarget 2020 Jan 21;11(3):265-281. Epub 2020 Jan 21.

Novartis Institutes for Biomedical Research, Oncology Disease Area, Cambridge, Massachusetts, USA.

SHP2 mediates RAS activation downstream of multiple receptor tyrosine kinases (RTKs) and cancer cell lines dependent on RTKs are in general dependent on SHP2. Profiling of the allosteric SHP2 inhibitor SHP099 across cancer cell lines harboring various RTK dependencies reveals that FGFR-dependent cells are often insensitive to SHP099 when compared to EGFR-dependent cells. We find that FGFR-driven cells depend on SHP2 but exhibit resistance to SHP2 inhibitors and . Treatment of such models with SHP2 inhibitors results in an initial decrease in phosphorylated ERK1/2 (p-ERK) levels, however p-ERK levels rapidly rebound within two hours. This p-ERK rebound is blocked by FGFR inhibitors or high doses of SHP2 inhibitors. Mechanistically, compared with EGFR-driven cells, FGFR-driven cells tend to express high levels of RTK negative regulators such as the SPRY family proteins, which are rapidly downregulated upon ERK inhibition. Moreover, over-expression of SPRY4 in FGFR-driven cells prevents MAPK pathway reactivation and sensitizes them to SHP2 inhibitors. We also identified two novel combination approaches to enhance the efficacy of SHP2 inhibitors, either with a distinct site 2 allosteric SHP2 inhibitor or with a RAS-SOS1 interaction inhibitor. Our findings suggest the rapid FGFR feedback activation following initial pathway inhibition by SHP2 inhibitors may promote the open conformation of SHP2 and lead to resistance to SHP2 inhibitors. These findings may assist to refine patient selection and predict resistance mechanisms in the clinical development of SHP2 inhibitors and to suggest strategies for discovering SHP2 inhibitors that are more effective against upstream feedback activation.
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http://dx.doi.org/10.18632/oncotarget.27435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980623PMC
January 2020

PTEN Loss Mediates Clinical Cross-Resistance to CDK4/6 and PI3Kα Inhibitors in Breast Cancer.

Cancer Discov 2020 01 8;10(1):72-85. Epub 2019 Oct 8.

Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, Massachusetts.

The combination of CDK4/6 inhibitors with antiestrogen therapies significantly improves clinical outcomes in ER-positive advanced breast cancer. To identify mechanisms of acquired resistance, we analyzed serial biopsies and rapid autopsies from patients treated with the combination of the CDK4/6 inhibitor ribociclib with letrozole. This study revealed that some resistant tumors acquired RB loss, whereas other tumors lost PTEN expression at the time of progression. In breast cancer cells, ablation of , through increased AKT activation, was sufficient to promote resistance to CDK4/6 inhibition and . Mechanistically, loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Because loss also causes resistance to PI3Kα inhibitors, currently approved in the post-CDK4/6 setting, these findings provide critical insight into how this single genetic event may cause clinical cross-resistance to multiple targeted therapies in the same patient, with implications for optimal treatment-sequencing strategies. SIGNIFICANCE: Our analysis of serial biopsies uncovered RB and PTEN loss as mechanisms of acquired resistance to CDK4/6 inhibitors, utilized as first-line treatment for ER-positive advanced breast cancer. Importantly, these findings have near-term clinical relevance because loss also limits the efficacy of PI3Kα inhibitors currently approved in the post-CDK4/6 setting..
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http://dx.doi.org/10.1158/2159-8290.CD-18-0830DOI Listing
January 2020

Tumor Intrinsic Efficacy by SHP2 and RTK Inhibitors in KRAS-Mutant Cancers.

Mol Cancer Ther 2019 12 22;18(12):2368-2380. Epub 2019 Aug 22.

Disease Area Oncology, Novartis Institute of Biomedical Research, Cambridge, Massachusetts.

, an oncogene mutated in nearly one third of human cancers, remains a pharmacologic challenge for direct inhibition except for recent advances in selective inhibitors targeting the G12C variant. Here, we report that selective inhibition of the protein tyrosine phosphatase, SHP2, can impair the proliferation of KRAS-mutant cancer cells and using cell line xenografts and primary human tumors. , sensitivity of KRAS-mutant cells toward the allosteric SHP2 inhibitor, SHP099, is not apparent when cells are grown on plastic in 2D monolayer, but is revealed when cells are grown as 3D multicellular spheroids. This antitumor activity is also observed in mouse models. Interrogation of the MAPK pathway in SHP099-treated KRAS-mutant cancer models demonstrated similar modulation of p-ERK and DUSP6 transcripts in 2D, 3D, and , suggesting a MAPK pathway-dependent mechanism and possible non-MAPK pathway-dependent mechanisms in tumor cells or tumor microenvironment for the efficacy. For the KRAS MIAPaCa-2 model, we demonstrate that the efficacy is cancer cell intrinsic as there is minimal antiangiogenic activity by SHP099, and the effects of SHP099 is recapitulated by genetic depletion of SHP2 in cancer cells. Furthermore, we demonstrate that SHP099 efficacy in KRAS-mutant models can be recapitulated with RTK inhibitors, suggesting RTK activity is responsible for the SHP2 activation. Taken together, these data reveal that many KRAS-mutant cancers depend on upstream signaling from RTK and SHP2, and provide a new therapeutic framework for treating KRAS-mutant cancers with SHP2 inhibitors.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0170DOI Listing
December 2019

Targeting FGFR overcomes EMT-mediated resistance in EGFR mutant non-small cell lung cancer.

Oncogene 2019 09 19;38(37):6399-6413. Epub 2019 Jul 19.

Massachusetts General Hospital (MGH) Cancer Center, Charlestown, MA, USA.

Evolved resistance to tyrosine kinase inhibitor (TKI)-targeted therapies remains a major clinical challenge. In epidermal growth factor receptor (EGFR) mutant non-small-cell lung cancer (NSCLC), failure of EGFR TKIs can result from both genetic and epigenetic mechanisms of acquired drug resistance. Widespread reports of histologic and gene expression changes consistent with an epithelial-to-mesenchymal transition (EMT) have been associated with initially surviving drug-tolerant persister cells, which can seed bona fide genetic mechanisms of resistance to EGFR TKIs. While therapeutic approaches targeting fully resistant cells, such as those harboring an EGFR mutation, have been developed, a clinical strategy for preventing the emergence of persister cells remains elusive. Using mesenchymal cell lines derived from biopsies of patients who progressed on EGFR TKI as surrogates for persister populations, we performed whole-genome CRISPR screening and identified fibroblast growth factor receptor 1 (FGFR1) as the top target promoting survival of mesenchymal EGFR mutant cancers. Although numerous previous reports of FGFR signaling contributing to EGFR TKI resistance in vitro exist, the data have not yet been sufficiently compelling to instigate a clinical trial testing this hypothesis, nor has the role of FGFR in promoting the survival of persister cells been elucidated. In this study, we find that combining EGFR and FGFR inhibitors inhibited the survival and expansion of EGFR mutant drug-tolerant cells over long time periods, preventing the development of fully resistant cancers in multiple vitro models and in vivo. These results suggest that dual EGFR and FGFR blockade may be a promising clinical strategy for both preventing and overcoming EMT-associated acquired drug resistance and provide motivation for the clinical study of combined EGFR and FGFR inhibition in EGFR-mutated NSCLCs.
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http://dx.doi.org/10.1038/s41388-019-0887-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742540PMC
September 2019

Acquired Resistance of EGFR-Mutated Lung Cancer to Tyrosine Kinase Inhibitor Treatment Promotes PARP Inhibitor Sensitivity.

Cell Rep 2019 06;27(12):3422-3432.e4

Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. Electronic address:

Lung cancers with oncogenic mutations in the epidermal growth factor receptor (EGFR) invariably acquire resistance to tyrosine kinase inhibitor (TKI) treatment. Vulnerabilities of EGFR TKI-resistant cancer cells that could be therapeutically exploited are incompletely understood. Here, we describe a poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor-sensitive phenotype that is conferred by TKI treatment in vitro and in vivo and appears independent of any particular TKI resistance mechanism. We find that PARP-1 protects cells against cytotoxic reactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). Compared to TKI-naive cells, TKI-resistant cells exhibit signs of increased RAC1 activity. PARP-1 catalytic function is required for PARylation of RAC1 at evolutionarily conserved sites in TKI-resistant cells, which restricts NOX-mediated ROS production. Our data identify a role of PARP-1 in controlling ROS levels upon EGFR TKI treatment, with potentially broad implications for therapeutic targeting of the mechanisms that govern the survival of oncogene-driven cancer cells.
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http://dx.doi.org/10.1016/j.celrep.2019.05.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624074PMC
June 2019

SHP2 Inhibition Overcomes RTK-Mediated Pathway Reactivation in KRAS-Mutant Tumors Treated with MEK Inhibitors.

Mol Cancer Ther 2019 07 8;18(7):1323-1334. Epub 2019 May 8.

Novartis Institutes for BioMedical Research, Oncology Disease Area, Cambridge, Massachusetts.

FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with the MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTK) are also feedback-activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using an SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS-mutant cancer cells, to a less extent in those harboring the G13D variant, and involves several RTKs, including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS, at least for the G12C, G12D, and G12V variants, and wild-type KRAS can also contribute significantly to the feedback activation. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS-mutant cancer cell proliferation and These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0852DOI Listing
July 2019

Genetic mechanisms of target antigen loss in CAR19 therapy of acute lymphoblastic leukemia.

Nat Med 2018 10 1;24(10):1504-1506. Epub 2018 Oct 1.

Novartis Institutes for BioMedical Research, Cambridge, MA, USA.

We identified genetic mutations in CD19 and loss of heterozygosity at the time of CD19 relapse to chimeric antigen receptor (CAR) therapy. The mutations are present in the vast majority of resistant tumor cells and are predicted to lead to a truncated protein with a nonfunctional or absent transmembrane domain and consequently to a loss of surface antigen. This irreversible loss of CD19 advocates for an alternative targeting or combination CAR approach.
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http://dx.doi.org/10.1038/s41591-018-0146-zDOI Listing
October 2018

NOTCH1 Represses MCL-1 Levels in GSI-resistant T-ALL, Making them Susceptible to ABT-263.

Clin Cancer Res 2019 01 17;25(1):312-324. Epub 2018 Sep 17.

Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, Massachusetts.

Purpose: Effective targeted therapies are lacking for refractory and relapsed T-cell acute lymphoblastic leukemia (T-ALL). Suppression of the NOTCH pathway using gamma-secretase inhibitors (GSI) is toxic and clinically not effective. The goal of this study was to identify alternative therapeutic strategies for T-ALL.

Experimental Design: We performed a comprehensive analysis of our high-throughput drug screen across hundreds of human cell lines including 15 T-ALL models. We validated and further studied the top hit, navitoclax (ABT-263). We used multiple human T-ALL cell lines as well as primary patient samples, and performed both experiments and studies on patient-derived xenograft models.

Results: We found that T-ALL are hypersensitive to navitoclax, an inhibitor of BCL2 family of antiapoptotic proteins. Importantly, GSI-resistant T-ALL are also susceptible to navitoclax. Sensitivity to navitoclax is due to low levels of MCL-1 in T-ALL. We identify an unsuspected regulation of mTORC1 by the NOTCH pathway, resulting in increased MCL-1 upon GSI treatment. Finally, we show that pharmacologic inhibition of mTORC1 lowers MCL-1 levels and further sensitizes cells to navitoclax and leads to tumor regressions .

Conclusions: Our results support the development of navitoclax, as single agent and in combination with mTOR inhibitors, as a new therapeutic strategy for T-ALL, including in the setting of GSI resistance.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320296PMC
January 2019

Hyperactivation of MAPK Signaling Is Deleterious to RAS/RAF-mutant Melanoma.

Mol Cancer Res 2019 01 10;17(1):199-211. Epub 2018 Sep 10.

Oncology, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.

The most frequent genetic alterations in melanoma are gain-of-function (GOF) mutations in BRAF, which result in RAF-MEK-ERK signaling pathway addiction. Despite therapeutic success of RAF and MEK inhibitors in treating BRAF-mutant tumors, a major challenge is the inevitable emergence of drug resistance, which often involves reactivation of the MAPK pathway. Interestingly, resistant tumors are often sensitive to drug withdrawal, suggesting that hyperactivation of the MAPK pathway is not tolerated. To further characterize this phenomenon, isogenic models of inducible MAPK hyperactivation in BRAF melanoma cells were generated by overexpression of ERK2. Using this model system, supraphysiologic levels of MAPK signaling led to cell death, which was reversed by MAPK inhibition. Furthermore, complete tumor regression was observed in an ERK2-overexpressing xenograft model. To identify mediators of MAPK hyperactivation-induced cell death, a large-scale pooled shRNA screen was conducted, which revealed that only shRNAs against and rescued loss of cell viability. This suggested that no single downstream ERK2 effector was required, consistent with pleiotropic effects on multiple cellular stress pathways. Intriguingly, the detrimental effect of MAPK hyperactivation could be partially attributed to secreted factors, and more than 100 differentially secreted proteins were identified. The effect of ERK2 overexpression was highly context dependent, as RAS/RAF mutant but not RAS/RAF wild-type melanoma were sensitive to this perturbation. IMPLICATIONS: This vulnerability to MAPK hyperactivation raises the possibility of novel therapeutic approaches for RAS/RAF-mutant cancers.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0327DOI Listing
January 2019

Genomic correlates of response to immune checkpoint blockade in microsatellite-stable solid tumors.

Nat Genet 2018 09 27;50(9):1271-1281. Epub 2018 Aug 27.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Tumor mutational burden correlates with response to immune checkpoint blockade in multiple solid tumors, although in microsatellite-stable tumors this association is of uncertain clinical utility. Here we uniformly analyzed whole-exome sequencing (WES) of 249 tumors and matched normal tissue from patients with clinically annotated outcomes to immune checkpoint therapy, including radiographic response, across multiple cancer types to examine additional tumor genomic features that contribute to selective response. Our analyses identified genomic correlates of response beyond mutational burden, including somatic events in individual driver genes, certain global mutational signatures, and specific HLA-restricted neoantigens. However, these features were often interrelated, highlighting the complexity of identifying genetic driver events that generate an immunoresponsive tumor environment. This study lays a path forward in analyzing large clinical cohorts in an integrated and multifaceted manner to enhance the ability to discover clinically meaningful predictive features of response to immune checkpoint blockade.
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http://dx.doi.org/10.1038/s41588-018-0200-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6119118PMC
September 2018

Heterogeneity and Coexistence of T790M and T790 Wild-Type Resistant Subclones Drive Mixed Response to Third-Generation Epidermal Growth Factor Receptor Inhibitors in Lung Cancer.

JCO Precis Oncol 2018 16;2018. Epub 2018 Jul 16.

Massachusetts General Hospital, Boston.

Purpose: Third-generation epidermal growth factor receptor (EGFR) inhibitors like nazartinib are active against mutation-positive lung cancers with T790M-mediated acquired resistance to initial anti-EGFR treatment, but some patients have mixed responses.

Methods: Multiple serial tumor and liquid biopsies were obtained from two patients before, during, and after treatment with nazartinib. Next-generation sequencing and droplet digital polymerase chain reaction were performed to assess heterogeneity and clonal dynamics.

Results: We observed the simultaneous emergence of T790M-dependent and -independent clones in both patients. Serial plasma droplet digital polymerase chain reaction illustrated shifts in relative clonal abundance in response to various systemic therapies, confirming a molecular basis for the clinical mixed radiographic responses observed.

Conclusion: Heterogeneous responses to treatment targeting a solitary resistance mechanism can be explained by coexistent tumor subclones harboring distinct genetic signatures. Serial liquid biopsies offer an opportunity to monitor clonal dynamics and the emergence of resistance and may represent a useful tool to guide therapeutic strategies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097183PMC
http://dx.doi.org/10.1200/PO.17.00263DOI Listing
July 2018

Sequential ALK Inhibitors Can Select for Lorlatinib-Resistant Compound Mutations in ALK-Positive Lung Cancer.

Cancer Discov 2018 06 12;8(6):714-729. Epub 2018 Apr 12.

Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts.

The cornerstone of treatment for advanced ALK-positive lung cancer is sequential therapy with increasingly potent and selective ALK inhibitors. The third-generation ALK inhibitor lorlatinib has demonstrated clinical activity in patients who failed previous ALK inhibitors. To define the spectrum of mutations that confer lorlatinib resistance, we performed accelerated mutagenesis screening of Ba/F3 cells expressing EML4-ALK. Under comparable conditions, -ethyl--nitrosourea (ENU) mutagenesis generated numerous crizotinib-resistant but no lorlatinib-resistant clones harboring single mutations. In similar screens with EML4-ALK containing single resistance mutations, numerous lorlatinib-resistant clones emerged harboring compound mutations. To determine the clinical relevance of these mutations, we analyzed repeat biopsies from lorlatinib-resistant patients. Seven of 20 samples (35%) harbored compound mutations, including two identified in the ENU screen. Whole-exome sequencing in three cases confirmed the stepwise accumulation of mutations during sequential treatment. These results suggest that sequential ALK inhibitors can foster the emergence of compound mutations, identification of which is critical to informing drug design and developing effective therapeutic strategies. Treatment with sequential first-, second-, and third-generation ALK inhibitors can select for compound mutations that confer high-level resistance to ALK-targeted therapies. A more efficacious long-term strategy may be up-front treatment with a third-generation ALK inhibitor to prevent the emergence of on-target resistance. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-1256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984716PMC
June 2018

SHP2 inhibition restores sensitivity in ALK-rearranged non-small-cell lung cancer resistant to ALK inhibitors.

Nat Med 2018 05 5;24(4):512-517. Epub 2018 Mar 5.

Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, USA.

Most anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung tumors initially respond to small-molecule ALK inhibitors, but drug resistance often develops. Of tumors that develop resistance to highly potent second-generation ALK inhibitors, approximately half harbor resistance mutations in ALK, while the other half have other mechanisms underlying resistance. Members of the latter group often have activation of at least one of several different tyrosine kinases driving resistance. Such tumors are not expected to respond to lorlatinib-a third-generation inhibitor targeting ALK that is able to overcome all clinically identified resistant mutations in ALK-and further therapeutic options are limited. Herein, we deployed a shRNA screen of 1,000 genes in multiple ALK-inhibitor-resistant patient-derived cells (PDCs) to discover those that confer sensitivity to ALK inhibition. This approach identified SHP2, a nonreceptor protein tyrosine phosphatase, as a common targetable resistance node in multiple PDCs. SHP2 provides a parallel survival input downstream of multiple tyrosine kinases that promote resistance to ALK inhibitors. Treatment with SHP099, the recently discovered small-molecule inhibitor of SHP2, in combination with the ALK tyrosine kinase inhibitor (TKI) ceritinib halted the growth of resistant PDCs through preventing compensatory RAS and ERK1 and ERK2 (ERK1/2) reactivation. These findings suggest that combined ALK and SHP2 inhibition may be a promising therapeutic strategy for resistant cancers driven by several different ALK-independent mechanisms underlying resistance.
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http://dx.doi.org/10.1038/nm.4497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343825PMC
May 2018

Tracking the Evolution of Resistance to ALK Tyrosine Kinase Inhibitors through Longitudinal Analysis of Circulating Tumor DNA.

JCO Precis Oncol 2018 23;2018. Epub 2018 Jan 23.

Department of Medicine, Massachusetts General Hospital, Boston, MA.

Purpose: rearrangements predict for sensitivity to ALK tyrosine kinase inhibitors (TKIs). However, responses to ALK TKIs are generally short-lived. Serial molecular analysis is an informative strategy for identifying genetic mediators of resistance. Although multiple studies support the clinical benefits of repeat tissue sampling, the clinical utility of longitudinal circulating tumor DNA analysis has not been established in ALK-positive lung cancer.

Methods: Using a 566-gene hybrid-capture next-generation sequencing (NGS) assay, we performed longitudinal analysis of plasma specimens from 22 ALK-positive patients with acquired resistance to ALK TKIs to track the evolution of resistance during treatment. To determine tissue-plasma concordance, we compared plasma findings to results of repeat biopsies.

Results: At progression, we detected an fusion in plasma from 19 (86%) of 22 patients, and identified resistance mutations in plasma specimens from 11 (50%) patients. There was 100% agreement between tissue- and plasma-detected fusions. Among 16 cases where contemporaneous plasma and tissue specimens were available, we observed 100% concordance between mutation calls. mutations emerged and disappeared during treatment with sequential ALK TKIs, suggesting that plasma mutation profiles were dependent on the specific TKI administered. G1202R, the most frequent plasma mutation detected after progression on a second-generation TKI, was consistently suppressed during treatment with lorlatinib.

Conclusions: Plasma genotyping by NGS is an effective method for detecting fusions and mutations in patients progressing on ALK TKIs. The correlation between plasma mutations and response to distinct ALK TKIs highlights the potential for plasma analysis to guide selection of ALK-directed therapies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785105PMC
http://dx.doi.org/10.1200/PO.17.00160DOI Listing
January 2018

Primary Patient-Derived Cancer Cells and Their Potential for Personalized Cancer Patient Care.

Cell Rep 2017 Dec;21(11):3298-3309

Massachusetts General Hospital Cancer Center, Boston, MA 02129, USA. Electronic address:

Personalized cancer therapy is based on a patient's tumor lineage, histopathology, expression analyses, and/or tumor DNA or RNA analysis. Here, we aim to develop an in vitro functional assay of a patient's living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care, we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care.
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http://dx.doi.org/10.1016/j.celrep.2017.11.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5745232PMC
December 2017

Epithelial-to-Mesenchymal Transition Antagonizes Response to Targeted Therapies in Lung Cancer by Suppressing BIM.

Clin Cancer Res 2018 01 19;24(1):197-208. Epub 2017 Oct 19.

Philips Institute for Oral Health Research, VCU School of Dentistry and Massey Cancer Center, Richmond, Virginia.

Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it. We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in -mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance. We observed that mesenchymal -mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance "free" cellular BIM levels both led to resensitization of mesenchymal -mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to -mutant lung cancers, as it was also observed in -mutant lung cancers and large datasets, including different cancer subtypes. Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-1577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959009PMC
January 2018

Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway.

Nature 2017 07 5;547(7664):453-457. Epub 2017 Jul 5.

Broad Institute, 415 Main Street, Cambridge, Massachusetts 02142, USA.

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFβ-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.
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http://dx.doi.org/10.1038/nature23007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667900PMC
July 2017

The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation.

Sci Transl Med 2017 05;9(391)

Edwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital (MGH) and Harvard Medical School (HMS), Boston, MA 02114, USA.

Although targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models of breast cancer that faithfully recapitulate the disparate clinical responses in these microenvironments, we observed that brain metastases evade phosphatidylinositide 3-kinase (PI3K) inhibition despite drug accumulation in the brain lesions. In comparison to extracranial disease, we observed increased HER3 expression and phosphorylation in brain lesions. HER3 blockade overcame the resistance of -amplified and/or -mutant breast cancer brain metastases to PI3K inhibitors, resulting in marked tumor growth delay and improvement in mouse survival. These data provide a mechanistic basis for therapeutic resistance in the brain microenvironment and identify translatable treatment strategies for -amplified and/or -mutant breast cancer brain metastases.
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http://dx.doi.org/10.1126/scitranslmed.aal4682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5917603PMC
May 2017

Discovery and Optimization of HKT288, a Cadherin-6-Targeting ADC for the Treatment of Ovarian and Renal Cancers.

Cancer Discov 2017 09 19;7(9):1030-1045. Epub 2017 May 19.

Novartis Institutes for Biomedical Research, Cambridge, Massachusetts.

Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 () as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models , including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation. We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. .
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http://dx.doi.org/10.1158/2159-8290.CD-16-1414DOI Listing
September 2017

Prospects for combining targeted and conventional cancer therapy with immunotherapy.

Nat Rev Cancer 2017 05 24;17(5):286-301. Epub 2017 Mar 24.

Exploratory Immuno-Oncology, Novartis Institutes for BioMedical Research.

Over the past 25 years, research in cancer therapeutics has largely focused on two distinct lines of enquiry. In one approach, efforts to understand the underlying cell-autonomous, genetic drivers of tumorigenesis have led to the development of clinically important targeted agents that result in profound, but often not durable, tumour responses in genetically defined patient populations. In the second parallel approach, exploration of the mechanisms of protective tumour immunity has provided several therapeutic strategies - most notably the 'immune checkpoint' antibodies that reverse the negative regulators of T cell function - that accomplish durable clinical responses in subsets of patients with various tumour types. The integration of these potentially complementary research fields provides new opportunities to improve cancer treatments. Targeted and immune-based therapies have already transformed the standard-of-care for several malignancies. However, additional insights into the effects of targeted therapies, along with conventional chemotherapy and radiation therapy, on the induction of antitumour immunity will help to advance the design of combination strategies that increase the rate of complete and durable clinical response in patients.
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http://dx.doi.org/10.1038/nrc.2017.17DOI Listing
May 2017

KEAP1 loss modulates sensitivity to kinase targeted therapy in lung cancer.

Elife 2017 02 1;6. Epub 2017 Feb 1.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States.

Inhibitors that target the receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (MAPK) pathway have led to clinical responses in lung and other cancers, but some patients fail to respond and in those that do resistance inevitably occurs (Balak et al., 2006; Kosaka et al., 2006; Rudin et al., 2013; Wagle et al., 2011). To understand intrinsic and acquired resistance to inhibition of MAPK signaling, we performed CRISPR-Cas9 gene deletion screens in the setting of BRAF, MEK, EGFR, and ALK inhibition. Loss of , a negative regulator of NFE2L2/NRF2, modulated the response to BRAF, MEK, EGFR, and ALK inhibition in BRAF-, NRAS-, KRAS-, EGFR-, and ALK-mutant lung cancer cells. Treatment with inhibitors targeting the RTK/MAPK pathway increased reactive oxygen species (ROS) in cells with intact KEAP1, and loss of KEAP1 abrogated this increase. In addition, loss of KEAP1 altered cell metabolism to allow cells to proliferate in the absence of MAPK signaling. These observations suggest that alterations in the KEAP1/NRF2 pathway may promote survival in the presence of multiple inhibitors targeting the RTK/Ras/MAPK pathway.
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http://dx.doi.org/10.7554/eLife.18970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5305212PMC
February 2017

Programmed Cell Death Ligand (PD-L1) Expression in Stage II and III Lung Adenocarcinomas and Nodal Metastases.

J Thorac Oncol 2017 03 2;12(3):458-466. Epub 2016 Nov 2.

Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts; Department of Pathology, Harvard Medical School, Boston, Massachusetts. Electronic address:

Introduction: Programmed death ligand 1 (PD-L1) expression determined by immunohistochemistry (IHC) may serve as a predictive biomarker for anti-PD-1/PD-L1 therapies; however, little is known about intertumoral heterogeneity of PD-L1 expression determined by IHC in lung adenocarcinomas (ADCs), and there have been conflicting results on the prognostic role of PD-L1 expression in ADCs.

Methods: PD-L1 expression was evaluated in resected stage II and III ADCs by using various cutoffs and correlated with clinicopathologic parameters and survival. PD-L1 expression was also compared between the primary tumor and lymph node metastases.

Results: There were 109 study cases. PD-L1 expression was seen in 56 (51%), 43 (39%), and 19 (17%) when cutoffs of at least 1%, at least 5%, and at least 50%, respectively, were used. Abundant intratumoral CD8-positive T cells were a significant predictor of the expression in the primary tumor, with cutoffs of 1% and 5% (p < 0.001 for both) by multivariate analysis, whereas they were a nonsignificant predictor of the expression with a 50% cutoff (p = 0.076). PD-L1 expression was concordant between the primary tumor and nodal metastasis in most of the cases, but it was discrepant in up to 38%. The discrepancy was attributed in part to different predominant histologic patterns between the primary and metastatic tumors. In the entire cohort, PD-L1 expression with all cutoffs had no bearing on 5-year recurrence-free survival.

Conclusions: PD-L1 expression is associated with abundant intratumoral CD8-positive T cells in resected ADCs, suggesting a predictive role of PD-L1 expression in anti-PD-1/PD-L1 therapies; however, the possible intertumoral heterogeneity of PD-L1 expression raises a concern about selecting the most appropriate sample for PD-L1 IHC.
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http://dx.doi.org/10.1016/j.jtho.2016.10.015DOI Listing
March 2017

Programmed Cell Death Ligand 1 Expression in Resected Lung Adenocarcinomas: Association with Immune Microenvironment.

J Thorac Oncol 2016 11 24;11(11):1869-1878. Epub 2016 Aug 24.

Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts; Department of Pathology, Harvard Medical School, Boston, Massachusetts. Electronic address:

Introduction: Programmed cell death ligand 1 (PD-L1) expression on tumor cells can be upregulated via activation of CD8 cytotoxic T lymphocytes (CTLs) or the T helper cell (Th1) pathway, counterbalancing the CTL/Th1 microenvironment. However, PD-L1 expression in association with subtypes of tumor-associated lymphocytes and molecular alterations has not been well characterized in lung adenocarcinomas.

Methods: PD-L1 expression was evaluated in 261 resected lung adenocarcinomas using tissue microarrays and various scoring systems, and was correlated with clinicopathologic/molecular features, including the extent/subtype of tumor-associated lymphocytes (i.e., CD8, T-bet [Th1 transcription factor], and GATA3 [Th2 transcription factor]), and patient outcomes.

Results: PD-L1 expression was present in 129 (49%), 95 (36.5%), and 62 (24%) cases using cutoffs of ≥1%, ≥5%, and ≥50%, respectively, 98 (38%) by H score and 72 (28%) by immune score. PD-L1 expression was associated with abundant CD8 and/or T-bet tumor-infiltrating lymphocytes and EGFR wild-type, significant smoking history, and aggressive pathologic features. In addition, concurrent PD-L1 expression and abundant CD8 tumor-associated lymphocytes were seen in 25% of KRAS mutants or cases with no alterations by clinical molecular testing as opposed to only 7.4% of EGFR mutants. PD-L1 expression was significantly associated with decreased progression-free and overall survival rates by univariate analysis, but not by multivariate analysis.

Conclusion: PD-L1 expression in resected lung adenocarcinomas is frequently observed in the presence of CTL/Th1 microenvironment, in particular in those with KRAS mutations or no common molecular alterations, suggesting that blockade of the PD-1/PD-L1 axis may be a promising treatment strategy to reinstitute active immune response for at least a subset of such patient populations.
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http://dx.doi.org/10.1016/j.jtho.2016.08.134DOI Listing
November 2016

Acquired Resistance to First-Line Afatinib and the Challenges of Prearranged Progression Biopsies.

J Thorac Oncol 2016 11 20;11(11):2022-2026. Epub 2016 Aug 20.

Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts. Electronic address:

Objectives: The mechanisms of acquired resistance to the irreversible EGFR inhibitor afatinib are not well documented. We performed this prospective clinical trial to determine the prevalence of the mutation T790M in afatinib-resistant patients.

Methods: Eligible patients had EGFR mutations; they were tyrosine kinase inhibitor-naive and were treated with afatinib, 40 mg daily. At enrollment, patients consented to a future repeat biopsy at the time of acquired resistance.

Results: A total of 24 patients were enrolled. The objective response rate was 58% (95% confidence interval [CI]: 37-78) with a median progression-free survival of 11.4 months (95% CI: 5.9-13.7) and median overall survival of 20.8 months (95% CI: 15.1-40.5). Of the 24 patients enrolled, 23 progressed and only 14 completed repeat biopsy at time of progression, with 11 samples sufficient for molecular analysis. Of those 11 patients, four (36% [95% CI: 10.9-69.2]) harbored T790M.

Conclusions: T790M is likely a common resistance mechanism in patients treated with first-line afatinib. Although repeat biopsies at progression are crucial in elucidating resistance mechanisms, this study suggests that clinical and technical issues often limit their feasibility, highlighting the importance of developing noninvasive tumor-genotyping strategies.
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http://dx.doi.org/10.1016/j.jtho.2016.06.032DOI Listing
November 2016